Use of nrf2 inducers to treat epidermolysis bullosa simplex and related diseases

ABSTRACT

The present invention relates to methods and compositions for the prevention and treatment of keratin-based skin diseases. In particular, the application describes compositions and methods of treating a patient suffering from skin blistering comprising the use of phase II enzyme inducers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/929,985.

BACKGROUND OF THE INVENTION

The skin is continuously exposed to changes in the external environment,including oxidative insults, heat, cold, UV radiation, injury, andmechanical stresses. The stratum corneum, composed of terminallydifferentiated keratinocytes, constitutes the natural barrier thatprevents loss of water and prevents entry of infectious agents (e.g.,bacteria, viruses), small objects (e.g., particles), and a broad varietyof water-soluble chemicals.

Intermediate filaments (IFs), microtubules (MTs) and microfilaments(MFs) constitute the cytoskeleton and play important roles in theorganization and mechanical integrity of skin keratinocytes (Fuchs andCleveland, 1998). Keratins are a large family of proteins that form theintermediate filament cytoskeleton in epithelial cells. Keratins areencoded by two groups of genes, type I and II, which are distinct at thelevel of genomic structure and nucleotide sequence. Each type of keratingene is clustered within separate loci in the mouse and human genomes(Fuchs, 1995; Schweizer et al., 2006) Type II keratin proteins, whichinclude K1-K8/K71-K74 in soft epithelia and K81-K86 in hard epithelia,such as hair, nail, and oral papilla, are larger (52 to 70 kDa) andbasic-neutral in charge; type I keratins, which comprise K9-K28 in softepithelia and K31-K40 in hard epithelia, are smaller (40 to 64 kDa) andacidic (Schweizer et al., 2006). The physical proximity, identicalsubstructure and transcriptional orientation, and high sequence homologyof type II keratin genes K5, K6α, K6β and K6hf, and type I keratin genesK14, K16, K17 and K17n, strongly suggest that each subset was generatedthrough successive duplications from a common ancestral gene (Wong etal., 2005). Type I epidermal keratin genes K17, K16 and K17 share highamino acid sequence identity (Troyanovsky et al., 1992; McGowan andCoulombe, 1998a; 1998b), and are structurally and functionally related(Paladini and Coulombe, 1999; Coulombe et al., 2004; Tong and Coulombe,2006).

In most epithelial cells the keratin filament network spans the entirecytoplasm, from the surface of the nucleus to the cell periphery, whereit contacts cell-matrix (hemidesmosomes) and cell-cell (desmosomes)adhesive sites (e.g., Fuchs, 1995; Gu and Coulombe, 2007). Keratinintermediate filaments provide cells and tissues with mechanicalresilience and protects them against physical stress. Disruption of thekeratin scaffold leads to tissue and cell fragility in the skin and itsappendages (hair, nail, glands), oral mucosa, and cornea. Severalgenetic diseases are caused by dominantly-acting mutations altering thecoding sequence of keratin proteins (Fuchs and Cleveland, 1998; Gu andCoulomb; 2007; Irvine and McLean, 1999; Omary et al., 2004). Most ofthese mutations are missense or small in-frame insertions or deletionsaffecting the central rod domain of keratin proteins, and interferingwith their structural support function (Cassidy et al, 2002; Gu andCoulombe 2007).

Epidermolysis bullosa simplex (EBS) is a rare autosomal dominant diseasein which the epidermis loses its integrity following trivial mechanicaltrauma (Fine et al., 1991; 2000). The disease is characterized byextreme fragility of the keratinocytes, and skin blistering, resultingfrom missense mutations in the gene that encodes keratin 5 (K5) orkeratin 14 (K14) (Fuchs and Cleveland, 1998; Cassidy et al. 2002; Gu andCoulombe 2007; Omary et al., 2004). K5 and K14, which are abundantcellular proteins, normally co-polymerize to form an intricate networkof 10-12 nm-wide, “intermediate-sized” filaments in basal keratinocytesof epidermis and related epithelia (Nelson and Sun, 1983; Fuchs, 1995).EBS may manifest itself as a relatively mild blistering conditioninvolving the hands and feet (EBS, Weber-Cockayne type), or as ageneralized blistering condition, sometimes associated with mucosalblistering that involves the oropharynx, the esophagus and ocularmucosa, and which can be fatal (e.g., EBS, Dowling-Meara type). Inindividuals affected by EBS Weber-Cockayne (EBS-WC), blisters are rarelypresent at birth and may occur on the knees and shins with crawling, oron the feet in late infancy or later, during adolescence or earlyadulthood. Neonates affected by EBS, Koebner type (EBS-K), presentblisters at birth or develop blisters within the first few months oflife (Fine et al., 1991; 2000). In individuals suffering from EBS withmottled pigmentation (EBS-MP), skin fragility is evident at birth andchildren develop progressive brown pigmentation over time, interspersedwith depigmented spots on the trunk and extremities, which disappears inadult life (see Gu and Coulombe, 2007, and refs. therein). Individualsaffected by EBS-DM develop widespread and severe blistering and/ormultiple grouped clumps of small blisters at birth, with hyperkeratosisof the palms and soles, that improves during mid to late childhood. Theblistering in EBS-DM can be severe enough to result in neonatal orinfant death (Fine et al., 2000).

Management of all types of EBS consists of supportive care to protectthe skin from blistering, dressings that promotes healing, andprevention and treatment of secondary infection. These treatment optionsare therefore palliative and have limited success. Furthermore, EBS isrepresentative of a large number of tissue fragility conditions causedby inherited mutations in intermediate filament protein-encoding genes(see Fuchs and Cleveland, 1998; Cassidy et al., 2002; Omary et al.,2004; Gu and Coulombe, 2007).

Accordingly, there is a need in the art for improved treatment optionsfor EBS and the present invention satisfies that need.

SUMMARY OF THE INVENTION

It is, therefore, an object of the invention to provide solutions to theaforementioned deficiencies in the art.

Further to this object, the invention provides a method to amelioratethe compromised state of mechanical resilience of skin in a patientcomprising administering to the patient a composition comprising atherapeutically effective amount of an Nrf2 inducer. The patient to betreated may suffer from skin blistering. In one aspect of the invention,the patient may be affected by Epidermolysis bullosa simplex. In apreferred embodiment of the invention, the patient suffers fromEpidermolysis bullosa simplex-Weber-Cockayne type, Epidermolysis bullosasimplex-Koebner type, Epidermolysis bullosa simplex with mottledpigmentation, Epidermolysis bullosa simplex-Dowling-Meara type, orEpidermolysis bullosa simplex with muscular dystrophy.

In a further embodiment, the method to ameliorate the mechanicalresistance of the skin comprises administering a phase II enzymeinducer. In one embodiment, the phase II inducer is an isothiocyanate.In a preferred embodiment the phase TI enzyme inducer is sulforaphane.In another preferred embodiment, the phase II enzyme inducer is asulforaphane synthetic analogue. In yet another embodiment, the Nrf2inducer is keratinocyte growth factor, also known as fibroblast growthfactor 7. In an additional embodiment, the Nrf2 inducer is oltipraz. Ina further embodiment, the Nrf2 inducer is ethacrynic acid. In stillanother preferred embodiment, the Nrf2 inducer causes the selectiveinduction of K6, K16 or K17 in the keratinocytes in the skin of thepatient.

In an additional embodiment, the present invention provides a method fortreating or preventing skin blistering in a patient comprisingadministering to the patient a composition comprising a therapeuticallyeffective amount of an Nrf2 inducer. In one aspect of the invention, thepatient may be affected by Epidermolysis bullosa simplex. In a preferredembodiment of the invention, the patient suffers from Epidermolysisbullosa simplex-Weber-Cockayne type, Epidermolysis bullosasimplex-Koebner type, Epidermolysis bullosa simplex with mottledpigmentation, Epidermolysis bullosa simplex-Dowling-Meara type, orEpidermolysis bullosa simplex with muscular dystrophy.

In a further embodiment, the method for treating or preventing skinblistering comprises administering a phase II enzyme inducer. In oneembodiment, the phase II inducer is an isothiocyanate. In a preferredembodiment the phase II enzyme inducer is sulforaphane. In anotherpreferred embodiment, the phase II enzyme inducer is a sulforaphanesynthetic analogue. In yet another embodiment, the Nrf2 inducer iskeratinocyte growth factor. In an additional embodiment, the Nrf2inducer is oltipraz. In a further embodiment, the Nrf2 inducer isethacrynic acid. In a further embodiment, the Nrf2 inducer is a Michaelreaction acceptor, such as triterpenoids or cyclic/acyclicbis-benzylidene-alkalones. In still another preferred embodiment, theNrf2 inducer causes the selective induction of K6, K16 or K17 in thekeratinocytes in the skin of the patient.

In yet another embodiment, the present invention provides a method fortreating or preventing a keratin-based skin disease in a patientcomprising administering to the patient a composition comprising atherapeutically effective amount of an Nrf2 inducer. The patient to betreated may be affected by an epidermolytic or non-epidermolytickeratin-based skin disease. Exemplary types of keratin-based skindiseases (Cassidy et al., 2002) to be treated include, but are notlimited to, epidermolytic hyperkeratosis, ichtyosis bullosa of Siemens,pachyonychia congenita, epidermolytic or non-epidermolytic palmoplantarkeratoderma (diffuse or focal) steatocystoma multiplex,Naegeli-Franceschetti-Jadassohn syndrome and dermatopathia pigmentosareticularis. In one embodiment, the keratin-based skin disease is causedby a mutation at the K14 locus,

In a further embodiment, the method for treating or preventing akeratin-based skin disease comprises administering a phase II enzymeinducer. In one embodiment, the phase II inducer is an isothiocyanate.In a preferred embodiment the phase II enzyme inducer is sulforaphane.In another preferred embodiment, the phase II enzyme inducer is asulforaphane synthetic analogue. In yet another embodiment, the Nrf2inducer is keratinocyte growth factor. In an additional embodiment, theNrf2 inducer is oltipraz. In a further embodiment, the Nrf2 inducer isethacrynic acid. In a further embodiment, the Nrf2 inducer is a Michaelreaction acceptor, such as triterpenoids or cyclic/acyclicbis-benzylidene-alkalones. In still another preferred embodiment, theNrf2 inducer causes the selective induction of K6, K16 or K17 in thekeratinocytes in the skin of the patient.

In an additional embodiment, the present invention provides acomposition for topical application to the skin comprising atherapeutically effective amount of an Nrf2 inducer and a vehiclesuitable for delivery. Topical compositions may be in several forms,such as solutions, oils, creams, ointments, gels, lotions, or pastes,and include, for instance, the penetration enhancer “transcutanol”(diethylene glycol monoethylether), or other excipients well known inthe art.

Preferably, the Nrf2 inducer in the composition is a phase II enzymeinducer. More preferably, the phase II inducer is an isothiocyanate.Even more preferably, the phase II enzyme inducer is sulforaphane or asulforaphane synthetic analogue. In another embodiment, the Nrf2 induceris keratinocyte growth factor. In yet another embodiment, the Nrf2inducer is oltipraz. In a further embodiment, the Nrf2 inducer isethacrynic acid. In a further embodiment, the Nrf2 inducer is a Michaelreaction acceptor, such as triterpenoids or cyclic/acyclicbis-benzylidene-alkalones. In a preferred embodiment, the Nrf2 inducercauses the selective induction of K6, K16 or K17 in the keratinocytes inthe skin of the patient.

The Nrf2 inducer in the composition of the invention may be administeredalone or in combination with additional active agents, includingpharmaceutical, biological and/or molecular biological active agents inthe context of combination or adjuvant therapy.

The foregoing general description and following brief description of thedrawings and the detailed description are exemplary and explanatory and,along with the manuscript appended, are intended to provide furtherexplanation of the invention as claimed. Other objects, advantages, andnovel features will be readily apparent to those skilled in the art fromthe following detailed description of the invention.

BRIEF DESCRIPTION OF TYKE DRAWINGS

FIG. 1 illustrates the clinical features of Epidermolysis bullosasimplex in a 2-month old baby girl born prematurely. The patient hadrecurrent bullae from shortly after birth, particularly in trauma-proneareas. The blisters typically healed with hypopigmentation as newlesions formed. The patient's father bad similar lesions as a newbornand complained of recurrent bullae on his hands and feet,

FIG. 1A shows the presence of several skin erosions at various stages ofhealing in the abdomen and upper thighs (the source of frictional traumain the upper thighs is the diaper).

FIG. 1B details the presence of several large fluid-filled blisters onthe dorsal and lateral sides of toes and heel (depicted by single arrowsand double arrows, respectively). Some of the toenails are alsoaffected. The prominent soft bandage wrapped around the distal portionof the right foot is an attempt to prevent further trauma to the toes.

FIG. 2 illustrates the alignment of the predicted amino acid sequencesfor mouse K17 and human K17, K17 and K16. This alignment was producedusing the DNASIS v.3.5 software (Hitachi Software Engineering Inc.,Japan). Default parameters were applied. The boundaries of the majordomains recognized in all IF proteins (Fuchs and Weber, 1994) aredepicted with brackets: the non-helical head domain at the N-terminus;the α-helical subdomains 1A, 1B, 2A, and 2B characteristic of thecentral rod domain; and the non-helical tail domain at the C-terminus.Nonsense stop codons are depicted by asterisks. Both symbols “+” and “#”underneath the sequences identify residues that are different betweenmouse and human K17. The symbol “#” marks the subset for which mouse K17is identical to either K14 or K16; the symbol “̂” identifies residuesthat are conserved between mouse and human K17, but different from K14and K16.

DETAILED DESCRIPTION OF THE INVENTION

Transcription factor NF-E-related factor 2 (Nrf) belongs to the CNC(Cap-N-Collar) family of transcription factors and possesses a highlyconserved basic region-leucine zipper (blip) structure. Nrf2 plays acritical role in the constitutive and inducible expression ofanti-oxidant and detoxification genes, commonly known as phase II genes,that encode defensive enzymes, including drug metabolizing enzymes, suchas glutathione S-transferase, NADP(H):quinone oxidoreductase andUDP-glucuronosyltransferase, and anti-oxidant enzymes, such as hemeoxygenase-1-(HO-1)1 and γ-glutamylcysteine synthetase (GCS), in responseto oxidative and xenobiotic stress (Braun et al., 2002; Fahey et al.,1997; Fahey and Talalay, 1999; Holtzclaw et al., 2004; Motohashi andYamamoto, 2004). These enzymes are regulated through a promoter calledanti-oxidant responsive element (ARE) or electrophile response element(EpRE). Phase II genes are responsible for cellular defense mechanismsthat include the scavenging of reactive oxygen or nitrogen species (ROSor RNS), detoxification of electrophiles and maintenance ofintracellular reducing potential (Holtzclaw et al., 2004; Motohashi andYamamoto, 2004).

Nrf2 is normally sequestered in the cytoplasm of the cells by anactin-bound regulatory protein called Keap1. When cells are exposed tooxidative or electrophilic stress, the Keap1-Nrf2 complex undergoes aconformational change, and Nrf2 is liberated from the complex andreleased into the nucleus. The active Nrf2 dimerizes with small Mafproteins, binds to ARE and activates phase II gene transcription (Braunet al., 2002; Motohashi and Yamamoto, 2004).

There is increasing evidence that the induction of phase II enzymesprotects from carcinogenesis and mutagenesis and enhances theantioxidant capability of the cells (Fahey and Talalay, 1999; Iida etal., 2004). To date, nine classes of phase II enzyme inducers have beenidentified: 1) diphenols, phenylene diamincs and quinones; 2) Michaelacceptors; 3) isothiocyanates; 4) hydroperoxides and hydrogen peroxide;5) 1,2-dithiole-3-thiones; 6) dimercaptans; 7) trivalent arsenicals; 8)divalent heavy metals; and 9) carotenoids, curcumins and relatedpolyenes (Fahey and Talalay, 1999). These phase II enzyme inducers areconsidered very efficient antioxidants because unlike directantioxidants, they are not consumed stoichiometrically duringoxido-reduction reactions, have long duration of action, support thefunction of direct antioxidants, such as tocopherols and CoQ, andenhance the synthesis of glutathione, a strong antioxidant (Fahey andTalalay, 1999).

The diuretic ethacrynic acid (EA), an electrophilic Michael acceptor,oltipraz, and the isothiocyanate sulforaphane have been shown to inhibitlipopolysaccharide (LPS)-induced secretion of high-mobility group box 1(HMGB1), a proinflammatory protein implicated in the pathogenesis ofinflammatory diseases, from immunostimulated macrophages (Killeen etal., 2006). Oltipraz prevents carcinogenesis in liver and urinarybladder by enhancing carcinogen detoxification (Iida et al., 2004). Thecytoprotective effect of keratinocyte growth factor (KGF) againstoxidative stress in injured and inflamed tissues, including woundedskin, has been related to KGF's stimulation of Nrf2 during cutaneouswound repair (Braun et al., 2002).

Isothiocyanates, which are primarily derived from in calciferousvegetables, are potent antioxidants and effective agents in thechemoprevention of tumors via the activation of phase II enzymes,inhibition of carcinogen-activating phase I enzymes and induction ofapoptosis (Hecht, 1995; Zhang and Talalay, 1994; Zhang et al., 1994).Isothiocyanates are formed in plants from the hydrolysis ofglucosinolates, which are β-thioglucoside-N-hydroxysulfates, whenmaceration of the vegetables by predators, food preparation or chewingcauses disruption of the cells with consequent activation and release ofthe enzyme myrosinase. The resultant aglycones undergo non-enzymaticintramolecular rearrangement to yield isothiocyanates, nitriles andepithionitriles.

Sulforaphane is the aglycone breakdown product of the glucosinolateglucoraphanin, also known as sulforaphane glucosinolate (SGS). Themolecular formula of sulforaphane is C₆H₁₁NOS₂, and its molecular weightis 177.29 daltons. Sulforaphane is also known as 4-methylsulfinylbutylisothiocyanate and (−)-1-isothiocyanato-4(R)-(methylsulfinyl) butane.The structural formula of sulforaphane is:

Sulforaphane was first synthesized (Schmid and Karrer, 1948), and thenisolated from the weed hoary cress (Cardaria draba), savoy and redcabbage (Prochazka, 1959). More recently, sulforaphane was identified inbroccoli and shown to be a potent phase II enzyme inducer in isolatedmurine hepatoma cells (Zhang et al., 1992), block the formation ofmammary tumors in Sprague-Dawley rats (Zhang et al., 1994), preventpromotion of mouse skin tumorigenesis (Gills et al., 2006; Xu et al.,2006) and increase heme oxygenase-1 (HO-1) expression in human hepatomaHepG2 cells (Keum et al., 2006). Sulforaphane was also shown to inhibitultraviolet (UV) light-induced activation of the activator protein-1(AP-1), a promoter of skin carcinogenesis, in human keratinocytes (Zhuet al., 2004), and there is evidence that topical application ofsulforaphane extract increases the level of phase II enzymesNAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase A1and heme oxygenase 1 in mouse skin epidermis (Dinkova-Kostova et al.,2007). Moreover, sulforaphane protects human epidermal keratinocytesagainst sulfur mustard, a potent cytotoxic agent and powerful mutagenand carcinogen (Gross et al., 2006), and inhibits cell growth, activatesapoptosis, inhibits histone deacetylase (HDAC) activity and decreasesthe expression of estrogen receptor-α, epidermal growth factor receptorand human epidermal growth factor receptor-2, which are key proteinsinvolved in breast cancer proliferation, in human breast cancer cells(Pledgic-Tracy et al., 2007). Further, sulforaphane was showed toeradicate Helicobacter pylori from human gastric xenografts (Haristoy etal., 2003).

The present inventors discovered that topical application of Nrf2inducers to the skin markedly improve the mechanical resilience of skinand prevents or reduce skin blistering in mammals, and specifically inhuman subjects suffering from a keratin-based skin disease, particularlya skin disease caused by a mutation at the K14 locus, thanks to theirability to trigger ectopic expression of structurally- andfunctionally-related keratins in basal layer keratinocytes.

Type I epidermal keratins K14, K16, and K17 are remarkably similar atthe primary sequence level (see FIG. 2). There is direct and indirectevidence in the literature indicating that K14, K16, and K17 areredundant to a significant extent in their ability to foster structuralsupport in stratified epithelia such as epidermis. There also isevidence that K17 fulfills two additional functions, likely in acontext-dependent fashion, in skin epithelia. The first function is theprotection against TNFalpha-induced programmed cell death, which isshared with K14 and K16 (Tong and Coulombe, 2006). The second functionis stimulation of protein synthesis and epithelial cell growth, which isso far unique to K17 and restricted to the wound repair response (Kim etal., 2006) These two roles would be expected to be either neutral, orbeneficial, in the context of EBS treatment. It has also been proposedthat K16 plays a role in the process of keratinocyte activation thatoccurs after acute injury to the stratified epithelia (Paladini et al.,1996; Paladini and Coulombe, 1998; 1999). Since the structural supportfunction of keratins is defective in EBS and related conditions,accumulation of “surrogate” keratins (e.g., K16, K17) can “dilute away”,or attenuate, the dominant negative impact of the mutant protein (e.g.,K14) responsible for the disease (Cao et al., 2001; Kerns et al.).

The inventors of the present application have made the discovery thattreatment with sulforaphane (SF), a chemical naturally present in thediet, significantly decreases the massive skin blistering seen in amouse model of EBS, thanks to sulforaphane's ability to selectivelyinduce keratin genes whose structural support function is markedlyredundant with K14. These genes are K16 and K17 (see Paladini andCoulombe, 1999; McGowan et al., 2002; Coulombe et al., 2004; Tong andCoulombe, 2006) (FIG. 2). In contrast to its clear impact on K16 and K17expression, SF is not effective at inducing other relevant keratins inthe epidermis, including K5, or K14, and has a weaker impact on K6expression (Kerns et at.). The key aspect of SF's efficacy in treatingEBS consists in its ability to cause activation of K16 and K17expression in basal keratinocytes of epidermis. However, the mechanismby which sulforaphane causes induction of select keratins in treatedskins is not known.

Our studies have shown that sulforaphane's effect does not reach thebasal layer of epidermis, a key requirement for EBS therapy, whendissolved in an organic solvent like acetonitrile or acetone (date notshown). In contrast, sulforaphane impacts gene expression in the desiredmanner in basal keratinocytes of mouse epidermis when topicallyadministered in select formulations that comprise a carrier, such as,but not limited to, jojoba oil and evening primrose oil, that allowssulforaphane to reach basal keratinocytes in the epidermis. Theseformulations may be modified according to various factors affectinghuman skin, including the age of the subject being treated and the siteof treatment in the body.

The terms “subject” and “patient” are used interchangeably and are meantto refer to an animal. In a preferred aspect of the invention, thepatient is a mammal. In the most preferred aspect of the invention, themammal is a human. Other suitable subjects or patients include, but arenot limited to, laboratory animals, such as mouse, rat, rabbit or guineapigs, farm animals and domestic animals or pets.

An epidermolytic or a non-epidermolytic keratin-based skin disease, asused in the current context, should be obvious to the person skilled inthe art, and is meant to include any abnormality in the skin, where akeratin gene mutation is involved in the etiology of the disorder or isaffected by the disorder. Examples of epidermolytic or anon-epidermolytic keratin-based skin diseases for which the currentinvention could be used preferably include, but are not limited to,epidermolysis bullosa simplex, epidermolytic hyperkeratosis, ichtyosisbullosa of Siemens, pachyonychia congenita, epidermolytic ornon-epidermolytic palmoplantar keratoderma (diffuse or focal)steatocystoma multiplex, Naegeli-Franceschetti-Jadassohn syndrome anddermatopathia pigmentosa reticularis.

The treatment envisioned by the invention can be used for patients witha pre-existing condition, or for patients pre-disposed to akeratin-based skin disease. Additionally, the methods of the inventioncan be used to alleviate symptoms of a keratin-based skin disease inpatients, or as a preventative measure in patients. Finally, thetreatment envisioned could also be used as a complement to other agentsin the context of a combination or adjuvant therapy for administrationto a subject that is being treated with one or more conventional drugs.Such drugs can be administered concurrently with, prior to orsequentially with Nrf2 inducer or phase II enzyme inducer treatment.

As used herein, “a pharmaceutically effective amount” is intended tomean an amount effective to elicit a cellular response that isclinically significant.

The present invention relates to methods of preventing or treating EBSand other keratin-based skin diseases using phase II enzyme inducers asdescribed above.

Isothiocyanates are compounds containing the isothiocyanate (NCS) moietyand are easily identifiable by one of ordinary skill in the art. Anexample of an isothiocyanate includes, but is not limited tosulforaphane or its analogs. The description and preparation ofisothiocyanate analogs is described in United States Reissue Patent36,784, and is hereby incorporated by reference in its entirety. Thesulforaphane analogs used in the present invention include6-isothiocyanato-2-hexanone, exo-2-acetyl-6-isothiocyanatonorbornane,exo-2-isothiocyanato-6-methylsulfonylnorbornane,6-isothiocyanato-2-hexanol, 1-isothiocyanato-4-dimethylphosphonylbutane,exo-2-(1′-hydroxyethyl)-5-isothiocyanatonorbornane,exo-2-acetyl-5-isothiocyanatonorbornane,1-isothiocyanato-5-methylsulfonylpentane,cis-3-(methylsulfonyl)cyclohexylmethylisothiocyanate andtrans-3-(methylsulfonyl)cyclohexylmethylisothiocyanate.

Other compounds contemplated by the present invention includekeratinocyte growth factor (KGF), oltipraz, ethacrynic acid, and analogsthereof, as well a additional Michael reaction acceptors, such astriterpenoids or cyclic/acyclic bis-benzylidene-alkaloses.

The compounds used in the methods of the present invention can beformulated into pharmaceutical compositions with suitable,pharmaceutically acceptable excipients for topical administration tomammals. Such excipients are well known in the art. Topicaladministration includes administration to the skin or mucosa, includingsurfaces of the lung and eye.

Dosage forms for topical administration include, but are not limited to,ointments, creams, emulsions, lotions and gels and agents that favorpenetration within the epidermis. In a preferred embodiment, thecomposition is in the form of topical ointment.

The compounds of the invention may be administered alone or incombination with additional active agents, including pharmaceutical,biological and/or molecular biological active agents in the context ofcombination or adjuvant therapy. The compositions can also containadjuvants such as, but not limited to, solubilizers, skin permeationenhancers, preservatives, wetting agents, moisturizers, gelling agents,buffering agents, surfactants, emulsifying agents, emollients,thickening agents, stabilizers, humectants and dispersing agents.

Moisturizers include carriers that allow the Nrf2 inducer or phase IIenzyme inducer to reach basal keratinocytes in the epidermis. This maybe achieved by varying the formulation according to several factorsaffecting human skin, including the age of the subject being treated andthe body site. Examples of moisturizers include, but are not limited to,jojoba oil and evening primrose oil.

Suitable skin permeation enhancers are well known in the art and includelower alkanols, such as methanol ethanol and 2-propanol; alkyl methylsulfoxides such as dimethylsulfoxide (DMSO), decylmethylsulfoxide (C₁₀MSO) and tetradecylmethyl sulfoxide; pyrrolidones, urea;N,N-diethyl-m-toluamide; C₂-C₆ alkanediols; dimethyl formamide (DMF),N,N-dimethylacetamide (DMA) and tetrahydrofurfuryl alcohol.

Examples of solubilizers include, but are not limited to, hydrophilicethers such as diethylene glycol monoethyl ether (ethoxydiglycol,available commercially as Transcutol®) and diethylene glycol monoethylether oleate (available commercially as Softcutol®); polyoxy 35 castoroil, polyoxy 40 hydrogenated castor oil, polyethylene glycol (PEG),particularly low molecular weight PEGs, such as PEG 300 and PEG 400, andpolyethylene glycol derivatives such as PEG-8 caprylic/capric glycerides(available commercially as Labrasol®); alkyl methyl sulfoxides, such asDMSO; pyrrolidones, DMA, and mixtures thereof.

Prevention and/or treatment of infections can be achieved by theinclusion of antibiotics, as well as various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like, in the compositions of the invention.

One of ordinary skill will appreciate that effective amounts of theagents in the compositions used in the methods of the invention can bedetermined empirically. It will be understood that, when administered toa human patient, the total daily usage of the composition of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon a variety of factors:the type and degree of the response to be achieved; the activity of thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the duration of the treatment; drugs used incombination or coincidental with the method of the invention; and likefactors well known in the medical arts.

Typically, the amount of Nrf2 inducer in the composition topicallyadministered to the patient will be from about 100 nmol to about 1μmol/cm², and the composition will be applied directly on the skin overrelevant portions of the body of the patient two or three times a week,so as to prevent or minimize blistering resulting from frictionaltrauma.

EXAMPLES Source of Sulforaphane

Pure sulforaphane (SF) was used in our studies (Zhang et al., 1992).

Example 1 K14 Null and K5 Null Mouse Strains as Models for Very SevereEBS Disease

Introduction of null mutations at the K14 locus (Lloyd et al., 1995) andK5 locus (Peters et al., 2001) in mice essentially abrogate the keratinfilament network in basal keratinocytes in the epidermis, and rendersthe keratinocytes acutely fragile in the face of physiological levels ofmechanical trauma. The presence of small amounts of K15, a type Ikeratin related to K14, leaves a residual but wispy keratin filamentnetwork in basal keratinocytes of K14 null epidermis. Accordingly, K5null mice show more extensive skin blistering and die sooner (beforeP0.5) than K14 null mice (P2-P3 in K14 null mice). Thus, these two mousemodels represent very severe forms of the disease.

The K14 null mouse strain (Lloyd et al., 1995) has proven to be the moreuseful model for these studies. The selective fragility of epidermalbasal cells and the associated trauma-induced skin blistering seen inK14-null mice mimics EBS as seen in humans. This said, this mouse modelpresents unique challenges that axe relevant only to a small subset ofhuman EBS patients (FIG. 1). The main challenge is premature death,which is largely due to extensive oral blistering and its acute impacton the feeding, stamina, and growth of newborn pups. Beginning shortlyafter birth, K14 null mice become fragile, lethargic, and can bedistinguished from their “normal” littermates (K14^(+/+) or K14^(+/−)).By P, K14 null pups are significantly smaller, lack milk in theirbellies, and are unable to close their mouths, correlating with localswelling. At P0 and P, their lips and tongue exhibit very severeepithelial blistering. In our hands, the mean survival of the K14-nullmice is 2.5±0.35 days (n=14, p<0.01). Oral lesions are only seenoccasionally in newborns afflicted with EBS (Fine et al., 1991; see alsoFIG. 1B). The K14 null mouse strain thus provides a very stringent testfor the notion that treatment with SF could be effective in thetherapeutic management of skin blistering in EBS patients.

Example 2 Ectopic Expression of Gli2 Rescues Skin Blistering in K14 NullMice but not in K5 Null Mice

Keratin 17 is a direct target for the transcription factor Gli, apowerful terminal effector of hedgehog signaling pathways (Bianchi etal., 2005). In Gli2^(TG) transgenic mice (Grachtchouk et al., 2000),expression of the Gli2 coding sequence is controlled by the K5 genepromoter, thereby causing its accumulation in the basal layer ofepidermis. Gli2^(TG) mice appear normal at birth and in the daysthereafter, but they develop epidermal hyperplasia as young adults,which progresses to basal cell carcinoma by 2-3 months of age.Availability of Gli2^(TG) transgenic mice provided an opportunity toconduct a “proof of principle” experiment, whereby constitutiveexpression of Gli2^(TG) transgene in the setting of K14^(−/−) miceshould cause a stable upregulation of K17 in basal keratinocytes ofepidermis, and hence, rescue their oral and skin blistering. Conversely,the Gli2^(TG) transgene should not rescue the phenotype arising in K5null mice, given that Gli2 has a very modest impact on type II keratingene regulation in epidermis (see Kerns ee al).

Gli2^(TG) transgenic mice were thus mated with K14 null mice, and theresulting offsprings analyzed for readouts relevant to the K14 nullmutation. Unlike their K14^(−/−) littermates, the K14^(−/−) Gli2^(TG)mice were initially viable and showed normal skin, correlating with thepresence of K17 in basal cells. This was in contrast to K5^(−/−)Gli2^(TG) mice, which died shortly after birth, exactly as straight K5null mice did. These findings strongly suggest that rescue of anEBS-like condition can be achieved by exploiting functional redundancywithin the keratin multigene family, and the presence or activation of arelevant transcription factor in basal keratinocytes can ectopicallyinduce a keratin gene without affecting epidermal physiology.

Example 3 Sulforaphane Selectively Induces K16 and K17 in SkinKeratinocytes in Vitro and in Vivo

To evaluate the effect of sulforaphane (SF) on keratin genetranscription in vitro, a mouse keratinocyte line (308 cells) wasexposed to 1 μM SF in acetonitrile vehicle, and mRNA levels weremeasured at 12, 24, and 48 hours after treatment. Relative to vehicletreatment, SF-treated keratinocytes showed a significant increase in themRNA levels of NQO1, a well-established SF target, at all time points asexpected (Dinkova-Kostova, et al., 2006). Similarly to NQO1, K17 and K16mRNAs were each elevated ˜2.5 fold at 12 h after SF treatment, but theirinduction was shorter-lived and levels returned to baseline by 24 hours.No significant change was measured for K5, K6a, K6b, K14 and K15 mRNAlevels. Indirect immunofluorescence revealed an obvious induction ofK17, but not K14, at the protein level.

At higher doses and in some specific contexts, SF induces apoptosis, orprogrammed cell death (Gamet-Payrestre et al., 2000; Fimongnari et al.,2002; Misiewicz et al., 2003; Gingras, et al., 2004). However, in theexperiments described above, SF did not alter the intrinsically low rateof apoptosis seen in primary cultures of mouse keratinocytes, whenpresent at a 1 μM concentration in the culture medium. At higherconcentrations (5 μM), SF did alter the rate of apoptosis (16% incontrol treated-cells versus 94% in SF-treated cells). Thus, at a dosesufficient to alter keratin gene expression in a selective fashion (1μM), SF does not cause apoptosis in cultured primary mousekeratinocytes.

To uncover whether SF had a similar effect on keratin expression in vivoand after sustained treatment, SKH-1 hairless mice were topicallytreated with 1 μmole SF in jojoba oil, a vehicle that readily penetratesthe skin (El Laithy and El-Shaboury, 2002), twice a week for four weeks.Compared to vehicle-treated skin, SF-treated skin showed markedlyincreased K17 immunoreactivity that extended to the basal layer of theepidermis. K16 immunoreactivity was also increased, while K14 showed nochange. Total protein extracts were prepared from the dorsal skin ofthese mice and analyzed to confirm these alterations in keratinexpression. Relative to vehicle-treated samples, SF-treated samplesexhibited increased levels of K17 and K16, whereas the level of K14 wasunaltered. This sustained treatment regimen did not appear to affectskin morphology or alter the rate of apoptosis, in agreement with aprevious study involving topical SF treatment at a higher frequency overan 11-week period (Dinkova-Kostova et al., 2006).

To ascertain whether chronic topical application can result in systemicexposure, blood and liver samples were tested for levels of SF and itsmetabolites, collectively known as dithio-carbamates (OTCs) (Zhang etal., 1996; Ye et al., 2002). The levels of SF and DTCs in blood andliver homogenates were below the sensitivity of the assay. Liverhomogenates were also tested for NQO1 activity, and there was nosignificant difference between control groups (1150 mOD/min/mg inuntreated mice; 1053 mOD/min/mg in vehicle-treated mice) and SF-treatedmice (1161 mOD/min/mg). Taken together, these results suggest thatsystemic exposure is unlikely to occur in the context of the treatmentregimen used.

Example 4 Optimization of Sulforaphane Treatment Regime for K14 NullMice

The effectiveness of treatment with sulforaphane (SF) in preventing orreducing skin blistering was tested in K14 null mice. The treatmentregimen initially used entailed topical application of 1 μmol SF injojoba oil (100 μl volume) at P0, P1, and P3. This postnatal treatmentregimen reduced cutaneous blistering in several K14 null pups, but the“clinical success” achieved proved variable. Histological analysesrevealed that K14-null mice already exhibited a significant amount of“sub-clinical blistering” at birth, that is, prior to the first SFapplication. In other instances where K14 null mice were successfullyrescued, expression of the “rescue” keratin (1016, K17) began prior tobirth in basal keratinocytes of the epidermis (Paladini and Coulombe,1999). SF administered to pregnant mice crosses the placental barrier(Coulombe and Kerns, unpublished data; Noyan-Ashraf et al., 2006), andcould conceivably cause an induction of K16/K17 in fetal epidermis at aprenatal stage. Pregnant female mice were given an intraperitoneal (IP)injection of 5 μmol SF, and the embryos were retrieved and their skinand body were separately assayed for levels of SF and DTCs. Theseanalyses provided evidence that K17 is indeed induced in fetalepidermis. Based on these results, the treatment regimen was set asconsisting of 3 IP injections of 5 μmol SF administered to the motherevery other day during the week prior to delivery, followed by topicalapplication of 1 μmol SF (in 100 μl of jojoba oil) at P0, P1, P3 and P5post-birth.

These findings established that the SF treatment regimen can be modifiedto meet the “clinical demands” of the overall phenotype.

Example 5 Sulforaphane Treatment Reduces Cutaneous Blistering in K14Null Mice

The revised sulforaphane (SF) treatment regimen had a dramatic impact onthe appearance and integrity of K14 null mouse skin. K14 null pups couldno longer be identified based on their appearance and behavior at P0.5and even P2.5 At P4.5, many of the K14 null pups showed limitedblistering restricted to the front paws. During this early postnatalperiod, the difference between untreated and SF-treated K14 null pupswas indeed dramatic. Whereas nearly all of the untreated K14 null pupshad died by P3 (n=14), 90% of the SF-treated K14 null pups (n=26) werealive and thriving at P4.

Histological analyses were conducted with a special focus on forepaw,which consistently shows very severe skin blistering in K14 null mice.At P2.5, the skin was significantly protected in SF-treated micerelative to untreated K14 null controls. K14 null pups had markedly lesssub-clinical blistering of their forepaws, back skin, and snout thancontrol at P0. These data were confirmed by quantification of thesurface area of forepaw skin showing blistering in untreated andSF-treated K14 null mice.

Virtually all of the SF-treated K14-null mice developed progressivewasting beyond P4, and most of them died within a day or two. Unlikeskin, which remained largely blister-free, the lips and oral mucosashowed the telltale signs of severe blistering. The topical mode of SFdelivery during the postnatal phase of the treatment regimen was noteffective for maintenance of K16/K17 expression in the oral mucosa, andaccordingly this component of the K14 null phenotype was likelyresponsible for the demise of the mice beyond P4.

Example 6 The Therapeutic Benefit of Sulforaphane Correlates with K17Induction in Epidermis

Sulforaphane (SF) treatment regimen had no obvious impact on mouseepidermal architecture. To further probe into this issue, intact backskin tissue from P2 mice, SF-treated and untreated, was subjected toultrastructural and immunohistochemical analyses. Phenotypic rescuecorrelated with the presence of K17 antigens in the basal layer ofSF-treated epidermis. These results contrasted with untreated K14 nullskin, which showed a spotty distribution of K17 (and K16) restricted tothe suprabasal compartment. Early and late differentiation markers, suchas K1 and filaggrin, were completely normal in SF-treated K14-null mouseepidermis.

The K14, K16 and K17 genes and proteins are highly conserved betweenhuman and mouse at the level of sequence (FIG. 2), tissue distribution,and regulation (McGowan et al., 1998; Coulombe et al., 2004). Inparticular, these keratins' ability to provide structural support in theepidermis are similar (Paladini and Coulombe, 1999; Kerns et al.). Thus,the data collected from the K14 null mouse model for EBS are directlyapplicable to the skin of patients suffering from EBS as a result ofmutations at the K14 locus. While there are only a few reportsdescribing the equivalent of a K14 null mutation in the human population(Chan et al., 1994; Rugg et al., 1994; Jonkman et al., 1996;El-Ghalbzouri et al., 2003), the present inventors have providedevidence that the SF-dependent induction of K16 and K17 in the basalkeratinocytes of human epidermis alleviates the dominant negative impactof missense K14 alleles.

Accordingly, topical application of SF or an Nrf2 inducer is effectivein preventing skin blistering in the relevant subset of EBS patients.Studies conducted in our laboratory have shown that SF-induced K17protein is very long-lived in newborn as well as adult mouse epidermis(Bernot et al., 2005). The data provided above show that, in the mouse,there is no systemic exposure to SF or a pharmacologically activemetabolite in the context of the topical treatment regimen devised.These findings indicate that, beyond infancy, EBS patients will achievea significant preventive benefit from topical application of SF or anNrf2 inducer twice a week for most body sites and under most conditions.More frequent applications may be needed during the first few monthsafter birth and especially during the neonatal period, when for unknownreasons the EBS clinical symptoms are most pronounced (Fine et al.,1991; 2000).

SF-mediated induction of K16 and K17, along with its impact on theexpression or metabolic and anti-oxidant enzymes and proteins, is alsobeneficial in the treatment of other conditions in which the skinexhibits fragility as a result of a mutation in a gene encoding a keycytoskeletal component.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodification and variations of the invention provided they come withinthe scope of the appended claims and their equivalents.

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1. A method to ameliorate the mechanical resilience of skin in a patientin need thereof comprising administering to the patient a compositioncomprising a therapeutically effective amount of an Nrf2 inducer.
 2. Themethod of claim 1, wherein the patient suffers from skin blistering. 3.The method of claim 1, wherein the Nrf2 inducer is a phase II enzymeinducer.
 4. The method of claim 3, wherein the phase II inducer is anisothiocyanate.
 5. The method of claim 4, wherein the phase IT enzymeinducer is sulforaphane.
 6. The method of claim 4, wherein the phase IIenzyme inducer is a sulforaphane synthetic analogue.
 7. (canceled) 8.The method of claim 1, wherein the Nrf2 inducer is keratinocyte growthfactor. 9-11. (canceled)
 12. The method of claim 1, wherein thecomposition comprising the Nrf2 inducer is topically administered to thepatient. 13-15. (canceled)
 16. The method of claim 1, wherein thepatient is a mammal.
 17. The method of claim 16, wherein the mammal is ahuman.
 18. A method for treating or preventing skin blistering in apatient comprising administering to the patient a composition comprisinga therapeutically effective amount of an Nrf2 inducer.
 19. The method ofclaim 18, wherein the patient suffers from Epidermolysis bullosasimplex.
 20. The method of claim 18, wherein the patient suffers fromEpidermolysis bullosa simplex-Weber-Cockayne type, Epidermolysis bullosasimplex-Koebner type, Epidermolysis bullosa simplex with mottledpigmentation, Epidermolysis bullosa simplex-Dowling-Meara type, orEpidermolysis bullosa simplex with muscular dystrophy.
 21. The method ofclaim 20, wherein the Epidermolysis bullosa simplex is caused by amutation at the K14 locus.
 22. The method of claim 18, wherein the Nrf2inducer is a phase II enzyme inducer. 23-36. (canceled)
 37. A method fortreating or preventing a keratin-based skin disease in a patientcomprising administering to the patient a composition comprising atherapeutically effective amount of an Nrf2 inducer.
 38. The method ofclaim 37, wherein the keratin-based skin disease is selected from thegroup consisting of epidermolytic hyperkeratosis, ichtyosis bullosa ofSiemens, pachyonychia congenita, epidermolytic or non-epidermolyticpalmoplantar keratoderma (diffuse or focal) steatocystoma multiplex,Nacgeli-Franceschetti-Jadassohn syndrome and dermatopathia pigmentosareticularis. 39-54. (canceled)
 55. A composition for topical applicationto the skin comprising a therapeutically effective amount of an Nrf2inducer and a carrier that allows the Nrf2 inducer to reach basalkeratinocytes in the epidermis.
 56. (canceled)
 57. The composition ofclaim 55, wherein the Nrf2 inducer is a phase II enzyme inducer. 58-67.(canceled)